Bacillus subtilis var. natto increases the resistance of Caenorhabditis elegans to gram-positive bacteria.

AIMS: This study aimed to investigate the effect of Bacillus subtilis var. natto on the susceptibility of the model host, Caenorhabditis elegans, to bacterial infection. METHODS AND RESULTS: Caenorhabditis elegans worms were fed with a standard food consisting of Escherichia coli OP50 strain (control) or B. subtilis (natto) during their larval stage. The worms were then infected with pathogenic bacteria. We analyzed their survival time and RNA sequencing-based transcriptome. Upon infection with Staphylococcus aureus and Enterococcus faecalis, the survival time of B. subtilis (natto)-fed worms was longer than that of the control. Transcriptome analyses showed upregulation of genes associated with innate immunity and defense response to gram-positive bacteria in B. subtilis (natto)-fed worms. CONCLUSIONS: Bacillus subtilis (natto) conferred an increased resistance of C. elegans to gram-positive bacteria. Our findings provided insights into the molecular mechanisms underlying B. subtilis (natto)-regulated host immunity and emphasized its probiotic properties for preventing and alleviating infections caused by gram-positive bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first study to show that B. subtilis (natto) confers specific resistance against gram-positive bacteria.

No significant association between stable iodine intake and thyroid dysfunction in children after the Fukushima Nuclear Disaster: an observational study.

PURPOSE: Stable iodine prophylaxis helps prevent childhood thyroid cancer in nuclear emergencies; however, there is limited information on its effect on thyroid function. This study aimed to examine thyroid function and autoimmunity among children and adolescents that took stable iodine after the Fukushima Nuclear Disaster. METHODS: For this observational study, data were obtained from children and adolescents that underwent thyroid cancer screening at Hirata Central Hospital from April 2012 to March 2018. Participant characteristics, including possible hypothyroidism and hyperthyroidism, were compared between the prophylaxis and no-prophylaxis groups. Multivariable logistic regression models were used to assess for possible hypothyroidism, autoantibodies positive, and hyperthyroidism. RESULTS: A total of 1,225 participants with stable iodine prophylaxis and 3,946 without prophylaxis were enrolled. Of those participants, blood samples were available for 144 and 1,201 participants in the prophylaxis and no-prophylaxis groups, respectively. There were 17 (11.8%) and 146 cases (12.2%) of possible hypothyroidism or autoantibodies positive cases in the prophylaxis and no-prophylaxis groups, respectively, and there were no cases and 3 cases (0.2%) of possible hyperthyroidism in those two groups, respectively. Multivariable analysis for possible hypothyroidism revealed no association between stable iodine intake and possible hypothyroidism or autoantibodies positive [odds ratio 0.716 (95% confidence interval 0.399-1.284)] (p = 0.262). We did not perform multivariable analysis for hyperthyroidism due to the limited number of cases. CONCLUSION: Significant adverse effects of stable iodine intake on thyroid function were not observed among children and adolescents 7 years after the Fukushima Nuclear Disaster.

Left ventricular outflow tract pressure gradient changes after carvedilol-disopyramide cotherapy in a cat with hypertrophic obstructive cardiomyopathy.

Disopyramide reduces the left ventricular outflow tract (LVOT) pressure gradient and improves symptoms in humans with hypertrophic obstructive cardiomyopathy (HOCM). However, the efficacy of disopyramide in cats has not been reported. We treated a cat with HOCM with carvedilol and disopyramide cotherapy and monitored the changes in LVOT flow velocity and N-terminal pro B-type natriuretic peptide (NT-proBNP) concentration. A 10-month-old neutered male Norwegian Forest cat was referred with a moderate systolic cardiac murmur. Echocardiography revealed thickening of the left ventricular wall, systolic anterior motion of the mitral valve leaflets, and turbulent aortic flow in the LVOT at systole. The LVOT flow velocity was 5.6 m/s. The plasma NT-proBNP concentration exceeded 1,500 pmol/L. The cat was diagnosed with HOCM and the ß-blocker carvedilol was started and gradually increased to 0.30 mg/kg, bid. After 57 days, the LVOT flow velocity (4.8 m/s) and plasma NT-proBNP concentration (870 pmol/L) had decreased but remained elevated. Therefore, disopyramide was added at 5.4 mg/kg po bid and increased to 10.9 mg/kg po bid after 22 days. After 141 days of carvedilol and disopyramide treatment, the systolic anterior motion of the mitral valve leaflets had disappeared and the LVOT flow velocity and plasma NT-proBNP concentration had decreased to 0.7 m/s and 499 pmol/L, respectively. No adverse effect has been observed during the follow-up. Disopyramide might relieve feline LVOT obstruction after only partial response to a beta-blocker. Further large-scale studies are required to investigate the efficacy and safety of disopyramide use in cats with moderate to severe HOCM.

Construction of a recombinant vaccinia virus expressing Babesia gibsoni thrombospondin-related anonymous protein and evaluation of its immunogenicity in mice.

Previously, we have identified a gene encoding thrombospondin-related anonymous protein of Babesia gibsoni (BgTRAP), and have shown that the antisera raised against recombinant BgTRAP expressed in Escherichia coli inhibited the growth of parasites. In the present study, a recombinant vaccinia virus expressing the BgTRAP (VV/BgTRAP) was constructed. A specific band with a molecular mass of 80 kDa, which is similar to that of native BgTRAP on the merozoites of B. gibsoni, was detected in the supernatant of VV/ BgTRAP-infected RK13 cells. Mice inoculated with VV/BgTRAP produced a specific antiBgTRAP response. The antiserum against VV/BgTRAP showed reactivity against the native BgTRAP on parasites. These results indicated that the recombinant vaccinia virus expressing BgTRAP might be a vaccine candidate against canine B. gibsoni infection.

Construction of a recombinant vaccinia virus expressing Babesia gibsoni thrombospondin-related anonymous protein and evaluation of its immunogenicity in mice

@#Previously, we have identified a gene encoding thrombospondin-related anonymous protein of Babesia gibsoni (BgTRAP), and have shown that the antisera raised against recombinant BgTRAP expressed in Escherichia coli inhibited the growth of parasites. In the present study, a recombinant vaccinia virus expressing the BgTRAP (VV/BgTRAP) was constructed. A specific band with a molecular mass of 80 kDa, which is similar to that of native BgTRAP on the merozoites of B. gibsoni, was detected in the supernatant of VV/ BgTRAP-infected RK13 cells. Mice inoculated with VV/BgTRAP produced a specific antiBgTRAP response. The antiserum against VV/BgTRAP showed reactivity against the native BgTRAP on parasites. These results indicated that the recombinant vaccinia virus expressing BgTRAP might be a vaccine candidate against canine B. gibsoni infection.

Application of Perfusate With Human-Derived Oxygen Carrier Solution Under Subnormothermic Machine Perfusion for Donation After Cardiac Death Liver Grafts in Pigs.

Oxygenation is necessary for aerobic metabolism, which maintains adenosine triphosphate within the graft organ. In recent years, some studies have demonstrated that subnormothermic machine perfusion (SNMP) with hemoglobin-based oxygen carriers has the potential to improve oxygen metabolism. OBJECTIVE: The aim of this study was to evaluate the effectiveness of perfusate with human-derived hemoglobin vesicles (HbV) under SNMP in a pig model of donation after cardiac death. MATERIALS AND METHODS: In this study, pig livers were procured with a warm ischemic time of 60 minutes and were preserved in 3 groups for 240 minutes. The preservation conditions were as follows: 4°C cold storage (Group 1); SNMP with University of Wisconsin perfusate alone (Group 2); and SNMP (21°C) with University of Wisconsin solution and HbV (hemoglobin, 0.6 mg/dL) perfusate (Group 3). All livers were perfused for 120 minutes using pig autologous blood machine perfusion (reperfusion phase). We investigated the aspartate transaminase level and hemodynamics (portal vein resistance and oxygen consumption) in the preservation and reperfusion phases. A histologic study (hematoxylin-eosin staining) was performed after 240 minutes of preservation. RESULTS: The portal vein resistance of Group 3 was not increased in comparison with Group 2. During preservation, the oxygen consumption of Group 3 was higher than that of Group 2. However, the level of aspartate transaminase did not differ between Groups 2 and 3. CONCLUSION: The present study revealed that perfusate with HbV increased the oxygen consumption of the donor liver during SNMP.

Mechanism of increased microbial autofluorescence by heat treatment.

The total direct count (TDC) microbial enumeration method is rapid and suitable for analysing environmental samples containing numerous un-culturable micro-organisms. Conventional TDC methods require the addition of a fluorescent stain and are thus unsuitable for automatic monitoring. We unexpectedly found that heated micro-organisms emit strong autofluorescence. This study was conducted to determine how heating enhances the autofluorescence of bacteria and fungi and to evaluate whether the phenomenon could be exploited to develop a new TDC method. Bacterial autofluorescence was augmented by heating cells at 200°C. ELISA results indicated that levels of advanced glycation end products (AGEs) increased in heated microbes. Catechin, an inhibitor of the Maillard reaction, disrupted the intensification of autofluorescence. These results suggest that the enhanced autofluorescence is associated with the formation of AGEs and that the reaction could be utilized as alternative probe in TDC methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Autofluorescence of bacteria and fungi was prominently intensified by heat treatment at 200°C. This phenomenon was associated with advanced glycation end products formed in micro-organisms via the Maillard reaction. The fluorescence signal was strong enough to be utilized as an alternative probe for fluorescent dye in the total direct count method. This phenomenon could be incorporated in an automatic apparatus for microbial enumeration, as it does not require staining.

Critical role of Myc activation in mouse hepatocarcinogenesis induced by the activation of AKT and RAS pathways.

MYC activation at modest levels has been frequently found in hepatocellular carcinoma. However, its significance in hepatocarcinogenesis has remained obscure. Here we examined the role of Myc activation in mouse liver tumours induced by hepatocytic expression of myristoylated AKT (AKT) and/or mutant HRASV12 (HRAS) via transposon-mediated gene integration. AKT or HRAS alone required 5 months to induce liver tumours, whereas their combination generated hepatocellular carcinoma within 8 weeks. Co-introduction of AKT and HRAS induced lipid-laden preneoplastic cells that grew into nodules composed of tumour cells with or without intracytoplasmic lipid, with the latter being more proliferative and associated with spontaneous Myc expression. AKT/HRAS-induced tumorigenesis was almost completely abolished when MadMyc, a competitive Myc inhibitor, was expressed simultaneously. The Tet-On induction of MadMyc in preneoplastic cells significantly inhibited the progression of AKT/HRAS-induced tumours; its induction in transformed cells suppressed their proliferative activity with alterations in lipid metabolism and protein translation. Transposon-mediated Myc overexpression facilitated tumorigenesis by AKT or HRAS, and when it was co-introduced with AKT and HRAS, diffusely infiltrating tumours without lipid accumulation developed as early as 2 weeks. Examination of the dose-responses of Myc in the enhancement of AKT/HRAS-induced tumorigenesis revealed that a reduction to one-third retained enhancing effect but three-times greater introduction damped the process with increased apoptosis. Myc overexpression suppressed the mRNA expression of proteins involved in the synthesis of fatty acids, and when combined with HRAS introduction, it also suppressed the mRNA expression of proteins involved in their degradation. Finally, the MYC-positive human hepatocellular carcinoma was characterized by the cytoplasm devoid of lipid accumulation, prominent nucleoli and a higher proliferative activity. Our results demonstrate that in hepatocarcinogenesis induced by both activated AKT and HRAS, activation of endogenous Myc is an enhancing factor and adequate levels of Myc deregulation further facilitate the process with alterations in cellular metabolism.

Comparison by multilocus variable-number tandem repeat analysis and antimicrobial resistance among atypical enteropathogenic Escherichia coli strains isolated from food samples and human and animal faecal specimens.

AIM: This study assessed whether multilocus variable-number tandem repeat analysis (MLVA) and antimicrobial susceptibility testing discriminated diarrhoeagenic atypical enteropathogenic Escherichia coli (aEPEC) from aEPEC indigenous to domestic animals or healthy people. METHODS AND RESULTS: MLVA genotyping of 142 aEPEC strains isolated from foods and faecal samples of domestic animals and humans revealed 126 distinct MLVA profiles that distributed to four clusters, yielding a Simpson's index of diversity (D) of 99·8%. Cluster 2 included 87% of cattle isolates and 67% of patient isolates. The plurality (15/34, 44%) of strains from healthy humans mapped to Cluster 1, while half (18/41, 44%) of the swine strains belonged to Cluster 4. Testing for antimicrobial susceptibility revealed that 52 strains (37%) of aEPEC were resistant to one or more agents; only 10 strains (7%) exhibited resistance to more than three agents. Strains isolated from swine or food exhibited a wider variety of resistance phenotypes than bovine or human strains. CONCLUSIONS: MLVA assigned the aEPEC isolates from cattle and patients to Cluster 2, distinct from aEPEC from other sources. Hog yards may be a larger source of drug-resistant strains than are cattle ranches. SIGNIFICANCE AND IMPACT OF THE STUDY: MLVA suggests that human diarrhoeagenic aEPEC are derived from cattle and are distinct from strains carried by healthy people and other animals. Cattle appear to be reservoirs of human diarrhoeagenic aEPEC.

Molecular characteristics of Clostridium difficile isolates from human and animals in the North Eastern region of India.

A total of 1034 samples were collected from different sources and C. difficile was isolated from 18 (9.04%) of 199 human, 9 (4.89%) of 184 cattle, 29 (12.44%) of 233 pig, and from 23 (13.94%) of 165 poultry samples. Variations were observed on the rate of isolation according to age and clinical conditions (diarrhoea). None of the samples from cow, sheep, goat, local chicken, and wild animals yielded any C. difficile. Out of those isolates, 8, 2, 19 and 6 isolates from human, cattle, pig and poultry, respectively were toxigenic. The toxigenic isolates carried both tcdA, and tcdB (A+B+) and most of the human and the pig isolates were also positive for binary toxin genes (cdtA and cdtB). The A+B+ isolates belonged to three different toxinotypes (0, VI and XXXIII). Human and pig A+B+ isolates belonged to three (045, 126 and ACD 019) and four (046, 087, 126 and ACD 011) different ribotypes, respectively and the ribotypes of two cattle isolates were 014 and ACD 010. Six A+B+ avian isolates belonged to six different ribotypes (014, 087, SLO 134, SLO 160, ACD 012, ACD 014). The non-toxigenic isolates from human, cattle, pig and poultry were grouped into 7, 4, 4 and 7 different ribotypes, respectively. PFGE analysis could not differentiate similar ribotypes/toxinotypes of toxigenic isolates. All the toxigenic isolates showed cytopathic effect on Vero and Hela cell monolayers at 1:100 dilutions of cell-free culture supernatants within 18-20 h of inoculation.

Applicability of Combined Use of Extracorporeal Support and Temperature-Controlled Machine Perfusion Preservation for Liver Procurement of Donors After Cardiac Death in Pigs.

BACKGROUND: The use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. The objective of this study is to determine the benefits of extracorporeal membrane oxygenation (ECMO) and subnormothermic machine perfusion (MP) with rewarming in a large animal model of DCD liver. METHODS: After cardiac arrest, the abdominal aorta and the inferior vena cava were cannulated and connected to an ECMO circuit. Porcine livers were perfused in situ with ECMO at 22°C for 60 minutes after 60 minutes of cardiac death. Then the livers were perfused for 4 hours by MP as a graft viability test. In group 1, non-in situ ECMO and grafts were preserved hypothermic MP. In group 2, non-in situ ECMO and grafts were preserved subnormothermic rewarming MP. In group 3, we used ECMO and subnormothermic rewarming MP. To assess potential methods and effect, effluent enzymes were measured. Portal vein and hepatic artery pressure during MP were evaluated. RESULTS: Effluent enzyme of AST, alanine aminotransferase and LDH as viability markers were significantly low (aspartate aminotransferase, 2899, 2292, and 972 IU/L; alanine aminotransferase, 134, 140, and 72 IU/L; and lactate dehydrogenase, 4354, 4455, and 1855 IU/L in each group, respectively). Portal vein and hepatic artery pressure during preservation came down smoothly in group 3 compared with group 1. CONCLUSIONS: The combined use of in situ subnormothermic ECMO and machine preservation with rewarming is more essential for the recovery and resuscitating function of DCD liver grafts.

Subnormothermic Machine Perfusion Preservation With Rewarming for Donation After Cardiac Death Liver Grafts in Pigs.

BACKGROUND: The use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. However, the implementation of such a strategy requires the development of novel preservation methods to recover from changes owing to warm ischemia. The aim of this study was to evaluate the effectiveness of subnormothermic machine perfusion (MP) preservation with rewarming for porcine DCD liver grafts for transplantation. METHODS: Porcine livers were perfused with newly developed MP system. The livers were perfused for 4 hours with modified University of Wisconsin gluconate solution. Group 1 grafts were preserved with no warm ischemia time (WIT) and hypothermic MP (HMP) for 4 hours. Group 2 grafts were preserved with WIT 60 minutes and HMP for 4 hours. Group 3 grafts were preserved with WIT 60 minutes and rewarming up to 25°C by MP (RMP) for 4 hours. RESULTS: The aspartate aminotransferase and lactate dehydrogenase in the effluent maintained at lower level in group 3 compared with group 2. However, tissue ATP levels did not recover in groups 2 and 3. Histologically, the fatty degenerate and swelling of the hepatocyte was slightly seen in all groups. The normal structure of the hepatocellular cords, the bile duct and the sinusoid endothelium were preserved in all groups. CONCLUSIONS: Potentially, subnormothermic preservation with rewarming is expected to help the recovery of function for DCD liver grafts.

Overexpression of melanoma-associated antigen D4 is an independent prognostic factor in squamous cell carcinoma of the esophagus.

To pursue an urgently needed treatment target for esophageal cancer (EC), we investigated the function of the recently discovered melanoma-associated antigen (MAGE)-D4 in squamous cell EC. MAGE-D4 messenger RNA (mRNA) expression was analyzed in nine EC cell lines using quantitative reverse transcription polymerase chain reaction. In 65 surgical specimens of squamous cell EC with no prior neoadjuvant therapy, MAGE-D4 mRNA expression in EC tissues and corresponding normal tissues was analyzed and compared, and evaluated in terms of clinicopathological factors. In representative cases, MAGE-D4 protein distribution was analyzed immunohistochemically. The heterogeneity of MAGE-D4 mRNA expression was confirmed in EC cell lines by quantitative reverse transcription polymerase chain reaction. In surgical specimens, MAGE-D4 mRNA expression was significantly higher in EC tissues than in corresponding normal tissues (P < 0.001). Patients with the highest MAGE-D4 mRNA expression in EC tissues (top quartile, n = 17) had significantly shorter overall survival than patients with low expression (2-year survival: 44% and 73%, respectively, P = 0.006). Univariate analysis identified age (≥65 years), lymphatic involvement, and high MAGE-D4 mRNA expression as significant prognostic factors; high MAGE-D4 mRNA expression was also an independent prognostic factor in multivariable analysis (hazard ratio: 2.194; P = 0.039) and was significantly associated with Brinkman index (P = 0.008) and preoperative carcinoembryonic antigen level (P = 0.002). Immunohistochemical MAGE-D4b expression was consistent with MAGE-D4 mRNA profiling. Our results suggest that MAGE-D4 overexpression influences tumor progression, and MADE-D4 can be a prognostic marker and a potential molecular target in squamous cell EC.